Antimicrobial peptides are common weapons in the natural defense arsenal of many types of organisms, including mammals, birds, reptiles, insects, plants and many microorganisms. Naturally occurring antimicrobial peptides are unique sequences that are about 10 to 50 amino acids in length. They tend to be rich in basic amino acids (lysine and arginine) and thus cationic. They are also often amphipathic in nature (i.e., one part of the molecule is hydrophilic while the other part is hydrophobic).
Although widely studied, the mode of action of antimicrobial peptides remains the subject of scientific debate. In many cases, the data suggests that the amphipathic peptides organize to form pores or channels in membranes (Durell (1992)). In other experiments, the antimicrobial peptides appear to disrupt a membrane by forming a “carpet-like” association with the membrane (Gazit (1995)). Either mechanism disrupts and kills cells by causing membrane depolarization and the loss of essential cellular components.
Microbial selectivity stems from the difference between mammalian and microbial cells as to the lipid composition of the membranes. The outer leaflet of mammalian cell membranes is almost entirely composed of electrically neutral, zwitterionic phospholipids, mainly phosphatidylcholine, sphingomyelin and cholesterol. By contrast, bacterial membranes consist of mainly negatively charged phospholipids, such as phosphatidylglycerol and cardiolipin. Thus, bacterial cells are susceptible to the cationic antimicrobial peptides, while mammalian cells are not. There is also some evidence to suggest that cancer cells may also differ in their membrane components from normal mammalian cells, making tumor cells susceptible to antimicrobial peptides.
Antimicrobial peptides can also be expected to have efficacy against viruses, such as HIV, herpes simplex and cytomegalovirus. However, the mechanism differs slightly. A virus is generally immune to membrane-bursting mechanisms because of the outer protein coat, but several antimicrobial peptides have shown antiviral activity by either blocking fusion of the virus with the host cell wall (thereby preventing transmission of the genetic material into the host cell) or by inhibiting replication of the virus once the host cell wall has been breached.
In light of the widespread appearance of pathogens that are drug resistant, there is interest in using antimicrobial peptides as an alternative to typical small molecule drugs if they could be economically produced. However, a practical limitation to large-scale uses of antimicrobial peptides is that they are expensive to produce in mass quantities. For example, peptide synthesis is very costly because the peptides are of unique sequence. Each amino acid must be added to a growing peptide chain, usually with less than perfect efficiency. Thus, as chain length increases, yields decrease.
The recombinant production of proteins provides some advantages over solid phase synthesis, including sequence fidelity, convenience, low cost, and the ability to produce longer proteins. However, recombinant techniques cannot be universally applied, and the recombinant production of antimicrobial peptides is particularly difficult due to their tendency to kill a variety of host cells. Even when synthesized as inactive fusion proteins, the precursor must still be cleaved to liberate the active peptide and further purification is usually required. These additional steps increase the cost and decrease the yield of the recombinant protein.
Demegen, Inc. of Pittsburg, Pa. owns several peptides which are being developed for medical use. One is D2A21 (FAKKFAKKFKLKEAKKFAKLFAFAF) (SEQ. ID. No. 65) being developed under the trade name DEMEGEL.™ This unique antimicrobial peptide is an amphipathic α-helix peptide that uses groups of 4 and 3 amino acids in order to keep the polar and non-polar faces aligned (3.6 residues/turn). It is synthesized by traditional methods, one amino acid at a time.
D2A21 has activity against a variety of cell types, including T. vaginalis, C. trachomatis, and P. aeruginosa. Preliminary results have also established anti-tumor activity in a rat prostate adenocarcinoma model, improving the survival rates from 25% to 75% and not causing any significant toxicities. Although uncertain of the basis for this activity, it is suggested that tumor cell membranes are substantially different from those of normal cells and therefore more susceptible to lysis by antimicrobial peptides (Arlotti (2001)). Finally, D2A21 has also been shown to have activity against the herpes simplex virus (HSV). When mixed with a modified lipid octyl-glycerol, D2A21 was better than five other peptides (including magainins and defensins) against HSV.
Although very promising, peptides like D2A21 must be made one amino acid at a time, for a cost of about US $50–500/g. As another example, nisin is an antimicrobial peptide used in processed dairy products, which sells for approximately $6000/pound of active peptide.
An alternative approach is to design peptides that have a several-amino acid repeat unit. The short sequence of amino acids could be synthesized less expensively than a long peptide and the repeat unit oligomerized to reach the full peptide length. Recent efforts using this approach include U.S. Pat. No. 5,789,542 and Javadpour (1996). These references teach that 7 residue (7mer) repeat units, polymerized into 14 and 21 residue peptides, can form the basis for antimicrobial peptides. By using the 7mer, a “simulated” α-helix is made, complete with an 3.5 amino acids per turn. However, the 7mer is still quite expensive to synthesize, thus limiting this approach.
Desirably, a process would exist that could inexpensively produce peptides having comparable antimicrobial activity to unique peptides. More desirably, the antimicrobial peptides produced by such a process would not require adherence to the classical α-helix structure, so that small repeat units of fewer than 7 residues could be used to construct the final peptide. By virtue of their simplicity, the peptides would be inexpensive to make, yet have significant antimicrobial activity.